向蛋白质的丝氨酸/苏氨酸残基添加O-连接的β-N-乙酰葡糖胺(O-GlcNAc)是在所有多细胞生物体中发现的普遍存在的翻译后修饰。像磷酸化一样,O-GlcNAc糖基化(O-GlcNAc化)是可诱导的并且调节无数的生理和病理过程。然而,由于检测和定量修饰的困难,理解O-GlcNAcylation的不同功能通常是具有挑战性的。因此,研究O-GlcNAcylation的强大方法对于阐明其在单个蛋白质调节中的关键作用至关重要,复杂的细胞过程,和疾病。在这一章中,我们描述了一组化学酶标记方法,以(1)检测感兴趣的蛋白质上的O-GlcNAcylation,(2)监测O-GlcNAcylation的总水平及其对感兴趣的蛋白质的化学计量的变化,和(3)能够将O-GlcNAc映射到蛋白质内的特定丝氨酸/苏氨酸残基以促进功能研究。首先,我们概述了多用途突变半乳糖基转移酶(Y289LGalT)的表达和纯化程序。然后,我们描述了使用Y289LGalT修饰具有功能性手柄的O-GlcNAc残基,N-叠氮基乙酰半乳糖胺(GalNAz)。最后,我们讨论了铜催化的叠氮化物-炔环加成“点击”反应将各种含炔的化学探针连接到GalNAz的几种应用,并演示了O-GlcNAc修饰的蛋白质的这种官能化可以用于实现上述(1)-(3)。总的来说,这些方法,利用市售试剂和标准蛋白质分析工具,将有助于促进我们对O-GlcNAcylation的多种重要功能的理解。
The addition of O-linked β-N-acetylglucosamine (O-GlcNAc) to serine/threonine residues of proteins is a ubiquitous posttranslational modification found in all multicellular organisms. Like phosphorylation, O-GlcNAc glycosylation (O-GlcNAcylation) is inducible and regulates a myriad of physiological and pathological processes. However, understanding the diverse functions of O-GlcNAcylation is often challenging due to the difficulty of detecting and quantifying the modification. Thus, robust methods to
study O-GlcNAcylation are essential to elucidate its key roles in the regulation of individual proteins, complex cellular processes, and disease. In this chapter, we describe a set of chemoenzymatic labeling methods to (1) detect O-GlcNAcylation on proteins of interest, (2) monitor changes in both the total levels of O-GlcNAcylation and its stoichiometry on proteins of interest, and (3) enable mapping of O-GlcNAc to specific serine/threonine residues within proteins to facilitate functional studies. First, we outline a procedure for the expression and purification of a multiuse mutant galactosyltransferase enzyme (Y289L GalT). We then describe the use of Y289L GalT to modify O-GlcNAc residues with a functional handle, N-azidoacetylgalactosamine (GalNAz). Finally, we discuss several applications of the copper-catalyzed azide-alkyne cycloaddition \"click\" reaction to attach various alkyne-containing chemical probes to GalNAz and demonstrate how this functionalization of O-GlcNAc-modified proteins can be used to realize (1)-(3) above. Overall, these methods, which utilize commercially available reagents and standard protein analytical tools, will serve to advance our understanding of the diverse and important functions of O-GlcNAcylation.